Abstract

The assembly of clathrin/AP-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, AP-1 association to protein-free liposomes had been shown to require phosphoinositides, Arf1•GTP, and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed AP-1 stabilization on liposomes to depend on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 domain, but requires the WDLW motif interacting with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines colocalize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on the clathrin and adaptor interaction sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by coimmunoprecipitation after crosslinking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.